Macrophages are the first line of defense against Mycobacterium tuberculosis and other microorganisms that circumvent the mechanical defenses of the airways. When macrophages encounter particles, they ingest them in a portion of the plasma membrane and form an intracellular vesicle termed a phagosome. Simultaneous with ingestion, signal transduction pathways are triggered that activate the microbicidal systems of the macrophage. Most bacteria are killed when phagosome membranes fuse with lysosome membranes and lysosome contents gain access to the ingested bacteria. In contrast, M. tuberculosis survives in macrophages within phagosomes that do not fuse with lysosomes, and thereby avoid being killed. The overall goal of the proposed research is to determine the molecular mechanism whereby M tuberculosis avoids phagosome-lysosome fusion. We will test the hypothesis that M. tuberculosis specifically blocks a step in the signal transduction pathway that culminates in Ca2+-dependent phagosome-lysosome fusion. As increases in intracellular ionized calcium concentrations ([Ca2+]i) are proven to be essential for phagosome-lysosome fusion in human neutrophils, we will determine whether increases in [Ca2+]i occur when macrophages ingest M. tuberculosis. If not increases in [Ca2+]i occur, we will identify the most proximal step in the signal transduction pathway that is defective and will characterize the mechanism of inhibition of this step in detail. If increases in [Ca2+]i do occur when macrophages ingest M. tuberculosis, we will determine whether Ca2+- dependent effectors (annexins and calmodulin-dependent protein kinase II) are functional. If we find signal transduction is not directly affected by Mr. tuberculosis, we will test the alternative hypothesis that M. tuberculosis enters macrophages by a receptor-mediated pathway that does not normally culminate in phagosome-lysosome fusion. If this proves to be the case, we will characterize the receptor(s) involved. We will also characterize the mechanism of ingestion, to determine whether it more closely resembles phagocytosis or receptor-mediated endocytosis. The information obtained from the proposed studies will guide efforts to prevent persistent infection with M. tuberculosis by significantly enhancing our understanding of the microbe-macrophage interaction.